Transcription terminators are sequences in DNA that stop the progress of RNA polymerase and thereby reduce the transcription of downstream genes. Antiterminators increase downstream gene expression by modifying polymerase so that it no longer recognizes or responds to terminators. Nascent transcripts encoded by the two cis acting antitermination sites ("put" sites) of bacteriophage HK022 promote such a modification: an E. coli RNA polymerase molecule that transcribes a put site acquires an increased ability to transcribe through downstream transcription terminators. Other polymerase molecules in the cell are unaffected. Antitermination requires persistent association of nascent put RNA with the polymerase. Put suppresses both factor dependent and intrinsic terminators and has no apparent terminator specificity. Efficient antitermination depends on the structure of the put transcript and of a highly conserved Zn-finger located in beta', the largest subunit of RNA polymerase. No additional protein factor is required. Isolation and characterization of substitutions within the Zn finger provide good evidence that this domain of the proteins interacts directly and specifically with put RNA. Computer simulations, enzymatic probing, comparison of the two HK022 put sites, and extensive analysis of put mutants have provided clues about the details of put RNA structure and their importance for antitermination. Our evidence suggests that a newly synthesized put transcript of approximately 60 nt folds into a structure consisting of two stem-loops separated by an unpaired base. Randomization of segments of put shows that there is a strong preference for particular bases at about 5 positions and weaker preference at at about 9 additional positions. The bases that constitute seven base pairs can be changed provided that complementarity is maintained. Finally, we have identified 5 positions where base identity is unimportant. These results should allow us to identify new antiterminator sites in other organisms and to understand the role of different regions of the put structure. Temperate bacteriophages form a long term and mutually beneficial association with their bacterial hosts called lysogeny. Lysogeny is a major mechanism of horizontal gene transfer between bacterial families, and temperate phages are a reservoir of bacterial genes -- including virulence factors -- that allow their hosts to survive environmental changes. Many temperate bacteriophages lysogenize their hosts by inserting the their chromosomes into specific sites ("attB" or bacterial attachment sites) in the host chromosome. Insertion requires Integrase, a virus-encoded protein that catalyzes recombination between phage and bacterial attachment sites. Bacterial chromosomes contain many attBs, each specific to one or a few phages. Although different Integrases share a common catalytic mechanism and are related to each other in structure, proteins encoded by different phages typically recombine only their cognate sets of attachment sites. The existing relationships and distribution of Integrases and attachment sites imply that phages occasionally change their insertion specificity over an evolutionary time scale. Alteration of insertion specificity cannot occur in a single step because it requires alterations of both integrase and the multiple integrase binding regions within the attachment sites. In addition, efficient insertion requires sequence identity within the "overlap region", a short DNA segment that lies between the points of DNA strand exchange in the attachment sites, and the attachment sites of different phages typically have different overlap regions. We are using two closely related but functionally distinct sets of integrases and attachment sites as a tool to understand how changes in insertion specificity might occur. We showed that the site specificity of phage lambda Integrase can be changed to that of phage HK022 by replacement of five lambda residues with their HK022 counterparts. By themselves, two of the replacements relax specificity: the double mutant efficiently recombines both lambda and HK022 attachment sites. The other three replacements restrict specificity: the triple mutant recombines neither lambda nor HK022 attachment sites, but does recombine mutant lambda sites that can also be recombined by HK022 Integrase. To understand how these mutations alter specificity, we asked if they affected recombination of sites other than those of wild type lambda and HK022. When phage lambda infects a cell that lacks attB, lysogeny is infrequent, and, when it occurs, Integrase usually catalyzes insertion of the phage chromosome into one of 10-20 secondary attachment sites. These sites are equivalent to attB mutants. Phage containing either or both of the relaxing int mutations lysogenized a host lacking attB with about the same frequency as wild type. One of the relaxed mutants differed from and the other was similar to wild type in its preferences for particular secondary sites. We conclude that specificity relaxation by these mutations is sequence-dependent and narrow in scope. The three restricting int mutations, which were tested as a triple mutant, strongly reduced the frequency of insertion into all secondary sites, and therefore specificity restriction is broad in scope. Among the few insertion sites that were found, one differed from any used by wild type phage or the relaxed mutants. A phage that contained all five mutations inserted into secondary sites about twice as frequently as wild type and had new preferences for individual sites. The preferences of the quintuple mutant were not a simple sum of the component mutations, suggesting that interaction among the protein domains affected by the replacements is an important element of specificity alteration. We used these findings to refine a model for the evolution of phage insertion specificity. The initial step is phage insertion into a secondary attachment site whose overlap region differs from that of the wild type attB. Abnormal excision of this phage from the bacterial chromosome replaces phage DNA with adjacent bacterial DNA and incorporates the new overlap region sequence into the phage attachment site. We propose that this phage variant is adapted to insert efficiently into the secondary host site by a series of relaxing and restricting int mutations. According to this model, new attachment sites evolve from sequences that already have some activity as Integrase substrates, an attractive feature not found in other models. In addition, the new phage attachment site will be located at one end of the bacterial DNA replacement, where it is usually found in existing phages.